Humans are extensively exposed to a wide variety of DNA alkylating agents (AAs) either directly, for example via cigarette smoke or indirectly via their formation in the gastrointestinal tract. Certain AAs, such as the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) are known human carcinogens but the role that other AAs may play in the aetiology of human cancers is still uncertain due to difficulties in assessing human exposure to these agents. The aim of this work was therefore to develop a screening method to assess exposure to AAs by measuring the formation of O6-alkylguanines (O6-AlkGs) in DNA and to apply it to an in vitro cell model and to the analysis of human DNA samples. To do this we took advantage of the Schizosaccharomyces pombe alkyltransferase-like protein (Atl1), which binds to all O6-AlkGs so far studied, to develop a slot blot assay (ASB assay). DNA standards containing different levels of O6-methylguanine (O6-MeG) were prepared using Temozolomide-treated calf thymus DNA (TMZ-CT DNA) and human placental DNA (TMZ-Plac DNA). Using 1 Âµg DNA, the limit of quantification and detection were approximately 1.0 and 0.5 fmoles O6-MeG/Î¼g DNA, respectively. Treatment of the TMZ-CT DNA standards with the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), reduced the detectable signal to background levels, confirming that the assay detected O6-MeG in TMZ-CT DNA. Importantly, CT DNA reacted in vitro with the AAs N-ethyl-N-nitrosourea, N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, N-benzyl-N-nitrosourea, N-allyl-N-nitrosourea and potassium diazoacetate, all produced strong signals in the assay demonstrating its ability to detect a range of different O6-AlkGs. In addition, a variety of human DNA samples have been analysed in this study: of 26 human DNA samples tested alongside TMZ-Plac DNA standards, nine had O6-AlkG levels in the range of 1.07-1.81 fmoles/Âµg DNA, ten had trace levels and in seven samples there was no detectable signal. No O6-AlkG was detected in all eleven human DNA samples tested with TMZ-CT DNA due to high signal background in the standards. In an in vitro study, MCF 10A cells were treated with up to 500 ïM TMZ and DNA damage measured using the ASB and neutral Comet assay and cytotoxicity by the MTT assay. TMZ was to some degree toxic in MCF 10A cells with 30-40% of cell growth inhibition at doses of 500 ÂµM of TMZ. 1.34 fmoles/Âµg DNA of O6-AlkG was detected in MCF 10A cells treated with the highest dose of TMZ (500 ÂµM). The neutral Comet assay showed that 350 and 500 ÂµM of TMZ generated a high level of DNA strand breaks and DNA fragmentation in MCF 10A cells. These data suggest that TMZ induced DNA damage in MCF 10A cells but had little effect on cell viability. Overall, these studies show the potential usefulness of this slot blot assay as a screening tool to detect and quantify total O6-alkGs in human DNA samples. To better understand the relationship between O6-alkGs and breast cancer risk, future work would require the investigation of larger numbers of human breast samples and a more sophisticated breast cancer model with a wider range of alkylating agents.